Proteins are typically identified through a series of distinct procedures. The protein(s) of interest are first separated and isolated, and then digested with an enzyme to create a mixture of peptides. The peptides are then analyzed using tandem mass spectrometry (MS/MS) to obtain a fragmentation pattern characteristic of the peptides. An identification procedure follows which generally employs database searching techniques to compare the fragmentation pattern data with other known patterns.
The enzyme most prevalently used in the digestion procedure is trypsin, which cleaves the protein on the C-terminal side of lysine or arginine residues. Trypsin is favored because lysine and arginine are present at about 11.7% of all peptide residues; consequently, peptides which emerge from digestion by trypsin typically contain between 6 and 25 amino acid residues, with an average of about ten amino acid residues. Peptides of this relatively short length have a molecular weight within a range that can be accommodated by most mass spectrometers. An additional benefit of trypsin digestion is that the presence of the basic lysine or arginine at the C-terminus in conjunction with the basic N-terminus yields doubly charged ions under the ionization conditions of ESI MS/MS analysis. A routine method to determine the amino acid sequence of these doubly-charged ions is to fragment them by “collision-induced dissociation” (CID) in a mass spectrometer. CID it typically performed by accelerating the peptide ions in a controlled fashion and forcing them to collide with inert gas molecules such as nitrogen or argon. As a result of one or more such collisions, these doubly-charged peptide ions have been found to fragment in a predictable way that yields useful information about the peptide in question. In particular, the presence of ‘b-ions’ and ‘y-ions’ in the CID fragment spectra is diagnostic enough to enable automated database searching algorithms to assign a peptide sequence with a high degree of confidence. It should be noted that other diagnostically useful peptide ions are produced as well by the CID process, such as a-, c-, x-, and z-ions. FIG. 4 illustrates various fragmentation ions of an example peptide including three alanine molecules. As shown, the peptide can fragment in different places along the peptide backbone. The am-xn-m fragments are created by a break between a carbon and the carboxyl bond, the bm-yn-m fragments are created by a break on the N-terminal side of amide bonds, while cm-zn-m fragments are created by a break on the C-terminal side of amide bonds in the backbone.
There are, however, classes of proteins that do not contain the average numbers of lysine or arginine residues. For example, membrane proteins include long spans of hydrophobic amino acid residues without lysine or arginine. When proteins that do not contain the average numbers of lysine or arginine residues are digested with trypsin, large sections of the insoluble protein having masses above the range of most mass spectrometers remain intact and thus cannot be identified by MS/MS.
While it is possible to use other enzymes or chemical digestion techniques for the proteins unamenable to trypsin digestion, peptides produced by cleavage by other enzymes or reagents generally do not have a basic lysine or arginine residue at the C-terminus and thus tend to produce singly-charged rather than doubly-charged precursor ions. Such singly-charged precursor ions do not produce the rich series of b-ions and y-ions and therefore tend to yield less useful sequence information when fragmented in a mass spectrometer.
It would therefore be of great use to provide a method of analyzing proteins normally indigestible using trypsin that can still yield the useful sequence information obtainable from trypsin-produced peptides.